In recent years, nucleic acids have acquired greater significance as therapeutically active substances, e.g. antisense RNAs and DNAs have proved to be effective agents for selectively inhibiting certain genetic sequences. Their mode of activity enables them to be used as therapeutic agents for blocking the expression of certain genes (such as deregulated oncogenes or viral genes) in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells and perform their inhibiting activity therein (Zamecnik et al., 1986), even though the intracellular concentration thereof is low, partly because of their restricted uptake through the cell membrane owing to the strong negative charge of the nucleic acids.
Another method of selectively inhibiting genes consists in the application of ribozymes. Here again there is the need to guarantee the highest possible concentration of active ribozymes in the cell, for which transportation into the cell is one of the limiting factors.
There is also a need for an efficient system for introducing nucleic acid into living cells in gene therapy. For this, genes are delivered into cells in order to achieve the synthesis of therapeutically active gene products in vivo.
Increasingly, there is a need for methods of treatment in which the therapeutically active DNA (or mRNA) is administered like a drug ("gene therapeutic agent") either once or repeatedly, as required. Examples of genetically caused diseases in which gene therapy constitutes a promising approach are hemophilia, betathalassemia and "Severe Combined Immune Deficiency" (SCID), a syndrome caused by a genetically induced lack of the enzyme adenosine deaminase. Other possible applications are in immune regulation in which a humoral or intracellular immunity is achieved by the administration of functional nucleic acid which codes for a secreted protein antigen or for a non-secreted protein antigen, by means of an inoculation. Other examples of genetic defects in which a nucleic acid coding for the defective gene can be administered, e.g. in a form tailored to the individual requirements, include muscular dystrophy (dystrophin gene), cystic fibrosis ("Cystic fibrosis transmembrane conductance regulator gene") and hypercholesterolemia (LDL receptor gene). Methods of treatment by gene therapy are also of potential significance where hormones, growth factors or proteins with a cytotoxic or immunomodulating activity are to be synthesized in the body.
Numerous solutions have already been proposed for improving the transportation of nucleic acids into living cells, which is one of the limiting factors in the therapeutic use thereof.
One of these possible solutions consists of directly modifying the nucleic acids, e.g. by substituting the charged phosphodiester groups with uncharged groups. Another possible method of direct modification consists in using nucleoside analogues. However, these proposals have various disadvantages, e.g. reduced binding to the target molecule, a poorer inhibitory effect and possible toxicity.
An alternative approach to the direct modification of the oligonucleotides consists in leaving the oligonucleotide per se unchanged and providing it with a group which gives it the desired properties, e.g. with molecules which facilitate transportation into the cells.
There are various known techniques for the genetic transformation of mammalian cells in vitro, but their use in vivo is restricted (they include the introduction of DNA by means of viruses, liposomes, electroporation, microinjection, cell fusion, DEAE-dextran or the calcium phosphate precipitation method).
Attempts have already been made to develop a soluble system which can be used in vivo to convey the DNA into the cells in targeted manner (Wu and Wu, 1987). This system was developed for hepatocytes and is based on the principle of coupling polylysine to a glycoprotein to which a receptor provided on the hepatocyte surface responds and then, by adding DNA, forming a soluble glycoprotein/polylysine/DNA complex which is taken up into the cell and, once taken up, allows the DNA sequence to be expressed.
This system is specific to hepatocytes and is defined, in terms of its function, by a relatively well characterized absorption mechanism involving the asialoglycoprotein receptor.
A broadly applicable and efficient transport system makes use of the transferrin receptor for taken up nucleic acids into the cell by means of transferrin-polycation conjugates. This system is the subject of EP-A1 0388 758, the contents of which are hereby referred to.
It has been shown that DNA transported into the cell by means of this system is expressed and that, if nucleic acid with an inhibiting effect is used, the inhibiting effect is not impaired by the transporting system. This system will hereinafter be referred to by the name "transferrinfection".
International Patent Application WO 91/17773 relates to a system for transporting nucleic acids with a specific effect for CD4-expressing cells. This system makes use of the receptor used by the HIV virus, by complexing the nucleic acid which is to be imported with a protein/polycation conjugate, the protein portion of which is a protein with the ability to bind to CD4, and bringing CD4-expressing cells into contact with the protein-polycation/nucleic acid complexes obtained. It has been demonstrated that DNA transported into the cell by means of this system (hereinafter referred to as CD4 transfection) is expressed in the cell.
What is common to both these inventions is the feature that they make use of specific cell functions to enable or facilitate the introduction of nucleic acid into the cell. In both cases, the absorption mechanisms proceed with the involvement of factors which are referred to for the purposes of the invention, as "internalizing factors". This means factors which bind to the cell surface and are internalized cell-type specifically, in the wider or narrower sense, possibly with the cooperation of other factors (e.g. cell surface proteins), and their internalization may be reversible or irreversible. The internalizing factor is conjugated with a substance of a polycationic nature which, because of its affinity for nucleic acids, establishes a connection between itself and the nucleic acid. (Substances with the ability to form a bond between nucleic acid and the internalizing factor are hereinafter referred to as "bonding factor".)
In the course of the two inventions which preceded the present invention it was found that the optimum introduction of nucleic acid into the cell could be achieved if the ratio of conjugate to nucleic acid was selected to be such that the internalizing factor-polycation/nucleic acid complexes were substantially electroneutral.